Total internal reflection microscopy method and device based on free-form surface shaping

Abstract

The invention discloses a total internal reflection microscopy device based on free-form surface shaping.The device comprises a light source and a line polarized light generating module, a free-form surface focusing module, a digital microscopy refection module, an optical enlargement transmission module and a fluorescence imaging module which are sequentially arranged along a light path, wherein the line polarized light generating module is used for converting light beams emitted by the light source into line polarized light; the free-form surface focusing module is used for converting the line polarized light into annular focused light; the digital microscopy refection module is used for selecting annular focused light reflected in an area; the optical enlargement transmission module is used for achieving total internal reflection illumination of the light beams; the fluorescence imaging module is used for exciting a sample to emit fluorescent light and collecting a fluorescent light signal image.The invention further discloses a total internal reflection microscopy method based on free-form surface shaping.According to the total internal reflection microscopy method and device based on free-form surface shaping, no mechanical vibration module is adopted, scanning is conducted more stably, and noise is lower; the utilization rate of laser energy is higher, and an imaging view field is more uniform; a DMD control controls scanning, the angle is more accurate, and it is helpful to achieve layered scanning and 3D image reconstruction.

Description

technical field [0001] The invention relates to the field of microscopic imaging, in particular to a total internal reflection microscopic method and device based on free-form surface shaping. Background technique [0002] With the development of biological science research, especially the study of membrane-related biological phenomena, there is a strong demand for a microscopic method that only observes the structure of single-layer samples. In the traditional microscopy method, the entire field of view is illuminated by the illumination beam in the z-axis direction during illumination, and the resolution and signal-to-noise ratio in the z-axis direction have not been high. For this reason, light sectioning microscope and total internal reflection microscope ( Tirf) are technical means to improve the z-axis resolution. [0003] The light section microscope adopts the method of lateral illumination, but due to the existence of the diffraction limit, the thinnest beam of lat...

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Problems solved by technology

However, due to the existence of laser speckle, the light field will be inhomogeneous

For this reason, the invention of a rotating total internal reflection microscope system (Ring-tirf) has evened the illumination light field by means of rotation, and there are two methods for realizing the rotation, such as the vibrating mirror proposed by the patent application number CN103597396A method and digital micromirror (DMD), but these two methods have their own defects. The galvanometer method cannot avoid angle vibration, and the method of digital micromirror will lead to a relatively low utilization rate of light energy, only about 5%.

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